Preparative Synthesis of dTDP-L-Rhamnose Through Combined Enzymatic Pathways

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy- D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4- keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6- dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1- phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identifiedbyHPLC, ESI-MS,andNMR,showingabout95%purity

One-Pot Multienzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production

A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-d-galactose, which was combined with LgtB, a β-(1,4) galactosyltransferase from <i>Neisseria meningitidis</i>, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-d-galactose by consuming two moles of acetylphosphate and one mole of d-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a d-galactose unit to a variety of sugar units such as d-glucose, rutinose, and 2-deoxy-d-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-d-galactose regeneration cycles (RC_max) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively